scholarly journals Bub1 regulates chromosome segregation in a kinetochore-independent manner

2009 ◽  
Vol 185 (5) ◽  
pp. 841-858 ◽  
Author(s):  
Christiane Klebig ◽  
Dirk Korinth ◽  
Patrick Meraldi
Keyword(s):  
Cell Lines ◽  
Chromosome Segregation ◽  
Spindle Checkpoint ◽  
Minor Role ◽  
Independent Manner ◽  
Lower Efficiency ◽  
Checkpoint Signaling ◽  
Chromosome Alignment ◽  
A Minor ◽  
Cancer Tissues

The kinetochore-bound protein kinase Bub1 performs two crucial functions during mitosis: it is essential for spindle checkpoint signaling and for correct chromosome alignment. Interestingly, Bub1 mutations are found in cancer tissues and cancer cell lines. Using an isogenic RNA interference complementation system in transformed HeLa cells and untransformed RPE1 cells, we investigate the effect of structural Bub1 mutants on chromosome segregation. We demonstrate that Bub1 regulates mitosis through the same mechanisms in both cell lines, suggesting a common regulatory network. Surprisingly, Bub1 can regulate chromosome segregation in a kinetochore-independent manner, albeit at lower efficiency. Its kinase activity is crucial for chromosome alignment but plays only a minor role in spindle checkpoint signaling. We also identify a novel conserved motif within Bub1 (amino acids 458–476) that is essential for spindle checkpoint signaling but does not regulate chromosome alignment, and we show that several cancer-related Bub1 mutants impair chromosome segregation, suggesting a possible link to tumorigenesis.

scholarly journals Haspin inhibitors reveal centromeric functions of Aurora B in chromosome segregation

2012 ◽  
Vol 199 (2) ◽  
pp. 251-268 ◽  
Author(s):  
Fangwei Wang ◽  
Natalia P. Ulyanova ◽  
John R. Daum ◽  
Debasis Patnaik ◽  
Anna V. Kateneva ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Metaphase Chromosome ◽  
Histone H3 ◽  
Spindle Checkpoint ◽  
Aurora B ◽  
Docking Site ◽  
Chromosome Movement ◽  
Checkpoint Signaling ◽  
Chromosome Alignment ◽  
Spindle Midzone

Haspin phosphorylates histone H3 at threonine-3 (H3T3ph), providing a docking site for the Aurora B complex at centromeres. Aurora B functions to correct improper kinetochore–microtubule attachments and alert the spindle checkpoint to the presence of misaligned chromosomes. We show that Haspin inhibitors decreased H3T3ph, resulting in loss of centromeric Aurora B and reduced phosphorylation of centromere and kinetochore Aurora B substrates. Consequently, metaphase chromosome alignment and spindle checkpoint signaling were compromised. These effects were phenocopied by microinjection of anti-H3T3ph antibodies. Retargeting Aurora B to centromeres partially restored checkpoint signaling and Aurora B–dependent phosphorylation at centromeres and kinetochores, bypassing the need for Haspin activity. Haspin inhibitors did not obviously affect phosphorylation of histone H3 at serine-10 (H3S10ph) by Aurora B on chromosome arms but, in Aurora B reactivation assays, recovery of H3S10ph was delayed. Haspin inhibitors did not block Aurora B localization to the spindle midzone in anaphase or Aurora B function in cytokinesis. Thus, Haspin inhibitors reveal centromeric roles of Aurora B in chromosome movement and spindle checkpoint signaling.

scholarly journals The yeast S phase checkpoint enables replicating chromosomes to bi-orient and restrain spindle extension during S phase distress

2005 ◽  
Vol 168 (7) ◽  
pp. 999-1012 ◽  
Author(s):  
Jeff Bachant ◽  
Shannon R. Jessen ◽  
Sarah E. Kavanaugh ◽  
Candida S. Fielding
Keyword(s):  
Dna Replication ◽  
Chromosome Segregation ◽  
Replication Fork ◽  
S Phase ◽  
Origin Of Replication ◽  
Independent Manner ◽  
Checkpoint Signaling ◽  
Hydroxyurea Treatment ◽  
Chromatid Cohesion ◽  
S Phase Checkpoint

The budding yeast S phase checkpoint responds to hydroxyurea-induced nucleotide depletion by preventing replication fork collapse and the segregation of unreplicated chromosomes. Although the block to chromosome segregation has been thought to occur by inhibiting anaphase, we show checkpoint-defective rad53 mutants undergo cycles of spindle extension and collapse after hydroxyurea treatment that are distinct from anaphase cells. Furthermore, chromatid cohesion, whose dissolution triggers anaphase, is dispensable for S phase checkpoint arrest. Kinetochore–spindle attachments are required to prevent spindle extension during replication blocks, and chromosomes with two centromeres or an origin of replication juxtaposed to a centromere rescue the rad53 checkpoint defect. These observations suggest that checkpoint signaling is required to generate an inward force involved in maintaining preanaphase spindle integrity during DNA replication distress. We propose that by promoting replication fork integrity under these conditions Rad53 ensures centromere duplication. Replicating chromosomes can then bi-orient in a cohesin-independent manner to restrain untimely spindle extension.

scholarly journals Shugoshin Promotes Sister Kinetochore Biorientation in Saccharomyces cerevisiae

2008 ◽  
Vol 19 (3) ◽  
pp. 1199-1209 ◽  
Author(s):  
Brendan M. Kiburz ◽  
Angelika Amon ◽  
Adele L. Marston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Minor Role ◽  
Meiotic Cell ◽  
Homologous Chromosomes ◽  
Cell Divisions ◽  
Sister Chromatids ◽  
Sister Kinetochore ◽  
A Minor ◽  
Meiosis I

Chromosome segregation must be executed accurately during both mitotic and meiotic cell divisions. Sgo1 plays a key role in ensuring faithful chromosome segregation in at least two ways. During meiosis this protein regulates the removal of cohesins, the proteins that hold sister chromatids together, from chromosomes. During mitosis, Sgo1 is required for sensing the absence of tension caused by sister kinetochores not being attached to microtubules emanating from opposite poles. Here we describe a differential requirement for Sgo1 in the segregation of homologous chromosomes and sister chromatids. Sgo1 plays only a minor role in segregating homologous chromosomes at meiosis I. In contrast, Sgo1 is important to bias sister kinetochores toward biorientation. We suggest that Sgo1 acts at sister kinetochores to promote their biorientation.

scholarly journals GTSE1 tunes microtubule stability for chromosome alignment and segregation through MCAK inhibition

2016 ◽  
Author(s):  
Shweta Bendre ◽  
Arnaud Rondelet ◽  
Conrad Hall ◽  
Nadine Schmidt ◽  
Yu-Chih Lin ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Chromosome Segregation ◽  
Genome Stability ◽  
Cancer Cell Lines ◽  
Dynamic Regulation ◽  
Spindle Positioning ◽  
Chromosome Missegregation ◽  
Microtubule Stability ◽  
Chromosome Alignment

AbstractThe dynamic regulation of microtubules during mitosis is critical for accurate chromosome segregation and genome stability. Cancer cell lines with hyperstabilized kinetochore microtubules have increased segregation errors and elevated chromosomal instability (CIN), but the genetic defects responsible remain largely unknown. The microtubule depolymerase MCAK can influence CIN through its impact on microtubule stability, but how its potent activity is controlled in cells remains unclear. Here we show that GTSE1, a protein found overexpressed in aneuploid cancer cell lines and tumours, regulates microtubule stability during mitosis by inhibiting MCAK microtubule depolymerase activity. Cells lacking GTSE1 have defects in chromosome alignment and spindle positioning due to microtubule instability caused by excess MCAK activity. Reducing GTSE1 levels in CIN cancer cell lines reduces chromosome missegregation defects, while artificially inducing GTSE1 levels in chromosomally stable cells elevates chromosome missegregation and CIN. Thus GTSE1 inhibition of MCAK activity regulates the balance of microtubule stability that determines the fidelity of chromosome alignment, segregation, and chromosomal stability.

scholarly journals GTSE1 tunes microtubule stability for chromosome alignment and segregation by inhibiting the microtubule depolymerase MCAK

2016 ◽  
Vol 215 (5) ◽  
pp. 631-647 ◽  
Author(s):  
Shweta Bendre ◽  
Arnaud Rondelet ◽  
Conrad Hall ◽  
Nadine Schmidt ◽  
Yu-Chih Lin ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Chromosome Segregation ◽  
Genome Stability ◽  
Cancer Cell Lines ◽  
Dynamic Regulation ◽  
Spindle Positioning ◽  
Chromosome Missegregation ◽  
Microtubule Stability ◽  
Chromosome Alignment

The dynamic regulation of microtubules (MTs) during mitosis is critical for accurate chromosome segregation and genome stability. Cancer cell lines with hyperstabilized kinetochore MTs have increased segregation errors and elevated chromosomal instability (CIN), but the genetic defects responsible remain largely unknown. The MT depolymerase MCAK (mitotic centromere-associated kinesin) can influence CIN through its impact on MT stability, but how its potent activity is controlled in cells remains unclear. In this study, we show that GTSE1, a protein found overexpressed in aneuploid cancer cell lines and tumors, regulates MT stability during mitosis by inhibiting MCAK MT depolymerase activity. Cells lacking GTSE1 have defects in chromosome alignment and spindle positioning as a result of MT instability caused by excess MCAK activity. Reducing GTSE1 levels in CIN cancer cell lines reduces chromosome missegregation defects, whereas artificially inducing GTSE1 levels in chromosomally stable cells elevates chromosome missegregation and CIN. Thus, GTSE1 inhibition of MCAK activity regulates the balance of MT stability that determines the fidelity of chromosome alignment, segregation, and chromosomal stability.

scholarly journals Kinetochore-localized BUB-1/BUB-3 complex promotes anaphase onset in C. elegans

2015 ◽  
Vol 209 (4) ◽  
pp. 507-517 ◽  
Author(s):  
Taekyung Kim ◽  
Mark W. Moyle ◽  
Pablo Lara-Gonzalez ◽  
Christian De Groot ◽  
Karen Oegema ◽  
...  
Keyword(s):  
Spindle Checkpoint ◽  
Kinase Domain ◽  
Mitotic Chromosomes ◽  
Checkpoint Signaling ◽  
C Elegans ◽  
Anaphase Onset ◽  
Checkpoint Pathway ◽  
Chromosome Alignment ◽  
Kinetochore Region

The conserved Bub1/Bub3 complex is recruited to the kinetochore region of mitotic chromosomes, where it initiates spindle checkpoint signaling and promotes chromosome alignment. Here we show that, in contrast to the expectation for a checkpoint pathway component, the BUB-1/BUB-3 complex promotes timely anaphase onset in Caenorhabditis elegans embryos. This activity of BUB-1/BUB-3 was independent of spindle checkpoint signaling but required kinetochore localization. BUB-1/BUB-3 inhibition equivalently delayed separase activation and other events occurring during mitotic exit. The anaphase promotion function required BUB-1’s kinase domain, but not its kinase activity, and this function was independent of the role of BUB-1/BUB-3 in chromosome alignment. These results reveal an unexpected role for the BUB-1/BUB-3 complex in promoting anaphase onset that is distinct from its well-studied functions in checkpoint signaling and chromosome alignment, and suggest a new mechanism contributing to the coordination of the metaphase-to-anaphase transition.

scholarly journals MCPH1 Lack of Function Enhances Mitotic Cell Sensitivity Caused by Catalytic Inhibitors of Topo II

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 406
Author(s):  
María Arroyo ◽  
Antonio Sánchez ◽  
Ana Cañuelo ◽  
Rosalía F. Heredia-Molina ◽  
Eduardo Martínez-Molina ◽  
...  
Keyword(s):  
Cell Death ◽  
Chromosome Segregation ◽  
Topoisomerase Ii ◽  
Mitotic Cell ◽  
Primary Microcephaly ◽  
Checkpoint Response ◽  
Checkpoint Pathway ◽  
Chromosome Alignment ◽  
Topo Ii ◽  
A Minor

The capacity of Topoisomerase II (Topo II) to remove DNA catenations that arise after replication is essential to ensure faithful chromosome segregation. Topo II activity is monitored during G2 by a specific checkpoint pathway that delays entry into mitosis until the chromosomes are properly decatenated. Recently, we demonstrated that the mitotic defects that are characteristic of cells depleted of MCPH1 function, a protein mutated in primary microcephaly, are not a consequence of a weakened G2 decatenation checkpoint response. However, the mitotic defects could be accounted for by a minor defect in the activity of Topo II during G2/M. To test this hypothesis, we have tracked at live single cell resolution the dynamics of mitosis in MCPH1 depleted HeLa cells upon catalytic inhibition of Topo II. Our analyses demonstrate that neither chromosome alignment nor segregation are more susceptible to minor perturbation in decatenation in MCPH1 deficient cells, as compared with control cells. Interestingly, MCPH1 depleted cells were more prone to mitotic cell death when decatenation was perturbed. Furthermore, when the G2 arrest that was induced by catalytic inhibition of Topo II was abrogated by Chk1 inhibition, the incidence of mitotic cell death was also increased. Taken together, our data suggest that the MCPH1 lack of function increases mitotic cell hypersensitivity to the catalytic inhibition of Topo II.

scholarly journals Phosphorylation of the Ndc80 complex protein, HEC1, by Nek2 kinase modulates chromosome alignment and signaling of the spindle assembly checkpoint

2011 ◽  
Vol 22 (19) ◽  
pp. 3584-3594 ◽  
Author(s):  
Randy Wei ◽  
Bryan Ngo ◽  
Guikai Wu ◽  
Wen-Hwa Lee
Keyword(s):  
Cell Death ◽  
Rna Interference ◽  
Molecular Mechanism ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Distribution Characteristic ◽  
Checkpoint Signaling ◽  
Chromosome Alignment ◽  
Ndc80 Complex ◽  
Complex Protein

The spindle assemble checkpoint (SAC) is critical for accurate chromosome segregation. Hec1 contributes to chromosome segregation in part by mediating SAC signaling and chromosome alignment. However, the molecular mechanism by which Hec1 modulates checkpoint signaling and alignment remains poorly understood. We found that Hec1 serine 165 (S165) is preferentially phosphorylated at kinetochores. Phosphorylated Hec1 serine 165 (pS165) specifically localized to kinetochores of misaligned chromosomes, showing a spatiotemporal distribution characteristic of SAC molecules. Expressing an RNA interference (RNAi)-resistant S165A mutant in Hec1-depleted cells permitted normal progression to metaphase, but accelerated the metaphase-to-anaphase transition. The S165A cells were defective in Mad1 and Mad2 localization to kinetochores, regardless of attachment status. These cells often entered anaphase with lagging chromosomes and elicited increased segregation errors and cell death. In contrast, expressing S165E mutant in Hec1-depleted cells triggered defective chromosome alignment and severe mitotic arrest associated with increased Mad1/Mad2 signals at prometaphase kinetochores. A small portion of S165E cells eventually bypassed the SAC but showed severe segregation errors. Nek2 is the primary kinase responsible for kinetochore pS165, while PP1 phosphatase may dephosphorylate pS165 during SAC silencing. Taken together, these results suggest that modifications of Hec1 S165 serve as an important mechanism in modulating SAC signaling and chromosome alignment.

scholarly journals Mutual antagonism of estrogen receptors alpha and beta and their preferred interactions with steroid receptor coactivators in human osteoblastic cell lines

2003 ◽  
Vol 176 (3) ◽  
pp. 349-357 ◽  
Author(s):  
DG Monroe ◽  
SA Johnsen ◽  
M Subramaniam ◽  
BJ Getz ◽  
S Khosla ◽  
...  
Keyword(s):  
Cell Lines ◽  
Transcriptional Activation ◽  
Cell Types ◽  
Minor Role ◽  
Osteoblastic Cell ◽  
Regulation Of Transcription ◽  
Estrogen Response ◽  
Mutual Antagonism ◽  
A Minor ◽  
Steroid Receptor Coactivators

Estrogen is a major sex steroid that affects the growth, maintenance, and homeostasis of the skeleton. Two isoforms of the estrogen receptor (ERalpha and ERbeta) mediate the transcriptional effects of estrogen. Although both isoforms of ER are present and functional in some human osteoblast (OB) cell lines, there is minimal information on the differential regulation of transcription by ERalpha and ERbeta homo- or heterodimers. This report demonstrates that ERalpha and ERbeta coexpression decreases the transcriptional capacity (relative to each ER isoform alone) on an estrogen response element-dependent reporter gene in OBs but not in other non-osteoblastic cell lines. These data suggest that ERalpha and ERbeta coexpression can differentially influence the degree of transcriptional activation in certain cell types. Interestingly, the overexpression of the steroid hormone receptor coactivator-1 (SRC1) resulted in preferential transcriptional enhancement by ERbeta as well as coexpressed ERalpha and ERbeta, whereas SRC2 overexpression appeared to preferentially enhance ERalpha transactivation. SRC3 overexpression failed to enhance estrogen-dependent transcription of any ER combination in OBs. Similar overexpression experiments in COS7 cells exhibited preferential enhancement of ERalpha function with all SRCs, including SRC3. Our data also demonstrated that SRC3 mRNA is reduced in osteoblastic cells, suggesting that SRC3 may have only a minor role in these cells. These data suggest that the transactivation capacity of various ER isoforms is both SRC species and cell type dependent.

scholarly journals Inhibitors of the Bub1 spindle assembly checkpoint kinase: synthesis of BAY-320 and comparison with 2OH-BNPP1

2021 ◽  
Vol 8 (12) ◽  
Author(s):  
Ilma Amalina ◽  
Ailsa Bennett ◽  
Helen Whalley ◽  
David Perera ◽  
Joanne C. McGrail ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Kinase Activity ◽  
Spindle Assembly ◽  
Minor Role ◽  
Serine Threonine Kinase ◽  
Chromosome Alignment ◽  
A Cell ◽  
A Minor ◽  
Target Effects

Bub1 is a serine/threonine kinase proposed to function centrally in mitotic chromosome alignment and the spindle assembly checkpoint (SAC); however, its role remains controversial. Although it is well documented that Bub1 phosphorylation of Histone 2A at T120 (H2ApT120) recruits Sgo1/2 to kinetochores, the requirement of its kinase activity for chromosome alignment and the SAC is debated. As small-molecule inhibitors are invaluable tools for investigating kinase function, we evaluated two potential Bub1 inhibitors: 2OH-BNPPI and BAY-320. After confirming that both inhibit Bub1 in vitro , we developed a cell-based assay for Bub1 inhibition. We overexpressed a fusion of Histone 2B and Bub1 kinase region, tethering it in proximity to H2A to generate a strong ectopic H2ApT120 signal along chromosome arms. Ectopic signal was effectively inhibited by BAY-320, but not 2OH-BNPP1 at concentrations tested. In addition, only BAY-320 was able to inhibit endogenous Bub1-mediated Sgo1 localization. Preliminary experiments using BAY-320 suggest a minor role for Bub1 kinase activity in chromosome alignment and the SAC; however, BAY-320 may exhibit off-target effects at the concentration required. Thus, 2OH-BNPP1 may not be an effective Bub1 inhibitor in cellulo , and while BAY-320 can inhibit Bub1 in cells, off-target effects highlight the need for improved Bub1 inhibitors.

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